Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations

Summary The ash-1 locus is in the proximal region of the left arm of the third chromosome of Drosophila melanogaster and the ash-2 locus is in the distal region of the right arm of the third chromosome. Mutations at either locus can cause homeotic transformations of the antenna to leg, proboscis to leg and/or antenna, dorsal prothorax to wing, first and third leg to second leg, haltere to wing, and genitalia to leg and/or antenna. Mutations at the ash-1 locus cause, in addition, transformations of the posterior wing and second leg to anterior wing and second leg, respectively. A similar spectrum of transformations is caused by mutations at yet another third chromosome locus, trithorax. One extraordinary aspect of mutations at all three of these loci is that they cause such a wide variety of transformations. For mutations at both of the loci that we have studied the expression of the homeotic phenotype is both disc-autonomous (as shown by injecting mutant discs into metamorphosing larvae) and cell autonomous (as shown by somatic recombination analysis). The original mutations which identified these two loci, although lethal, manifest variable expressivity and incomplete penetrance of the homeotic phenotype suggesting that they are hypomorphic. The phenotype of double mutants which were synthesized by combining different pairs of those original mutations manifest for two of the four pairs a greater degree of expressivity and slightly more penetrance of the homeotic transformations. This mutual enhancement suggests that the products of both loci interact in the same process. A third double mutant expresses a discless phenotype.Additional alleles have been recovered at both the ash-1 and the ash-2 loci. Some of these alleles as homozygotes or transheterozygotes express the wide range of transformations revealed first by double mutants. One of the alleles at the ash-1 locus when homozygous and several transheterozygous pairs can cause either the homeotic transformation of discs or the absence of those discs. The fact that these two defects, absence of specific discs and homeotic transformations of those same discs can be caused by mutations within a single gene suggests that the activity of the product of this gene is essential for normal imaginal disc cell proliferation. Loss of that activity leads to the absence of discs, whereas, reduction of that activity leads to homeotic transformations.     

A maternal effect mutation leading to deficiencies of organs and homeotic transformations in the adults ofDrosophila

Summary The temperature sensitive mutationfs(l)h is characterized at the restrictive temperature of 29°C by both a maternal effect responsible for the early embryonic lethality and pupal zygotic lethality. The two phenotypes are inseparable and map at a short deletion in the X chromosome (7Dl, 7D5-6). At semipermissive temperatures, hemizygous mutant females produce adults with morphological defects, such as organ deficiencies and homeotic transformations of haltere to wing and third leg to second leg. These defects depend on the maternal genotype and are governed by an early temperature sensitive period, which covers the end of oogenesis and the first hours of embryogenesis. Furthermore, this maternal effect mutation interacts with some dominant mutations of the bithorax system. These properties suggest thatfs(l)h is somehow involved in segmental determination.     

Temperature sensitivity ofTumorous head,a homoeotic mutant ofDrosophila melanogaster

Summary A temperature-sensitive period during early embryogenesis for three stocks carrying thetuh-3 gene suggests that it is a homoeotic mutation involved in the initial determination of the eye-antennal disc rather in maintenance of the determination.     

RFLP mapping of the barley homeotic mutant lax-a

The lax-a homeotic mutant of barley has flowers in which lodicules are replaced by stamens (giving five stamens per flower). RFLP mapping of an F₂ population from a Bonus lax-a ¹ x H. spontaneum cross showed that the mutation was on the short arm of chromosome 7(5H), closely linked to the centromere. An additional F₂ population was used to show that the lax-a mutation gave the five-stamen phenotype in all flowers of 6-rowed spikes and that hoods were elevated and reduced in size in lax-a/Hooded double-mutant plants.     

Transdetermination in leg imaginal discs ofDrosophila melanogaster undDrosophila nigromelanica

Summary The transdetermination capacities of leg discs ofDrosophila melanogaster were examined by mechanically disrupting and kneading whole discs from late third instar larvae and by culturing the resulting tissue mass for 10–14 days in adult female abdomens where the cells continued to divide. The grown implants were then dissected from the abdomens and injected into third instar larvae to undergo metamorphosis.After this treatment, prothoracic leg discs ofDrosophila melanogaster transdetermined with a high frequency (59% of all implants) to wing. Mesothoracic leg discs also transdetermined to wing, but at a very low frequency (4%). Metathoracic leg discs exhibited the same low frequency of transdetermination (4%), but in this case the direction of transdetermination was to haltere (Table 1,D. melanogaster).Very similar results were obtained with leg discs ofDrosophila nigromelanica (Table 1,D. nigromelanica), showing that the peculiar behavior of the three leg discs is not unique forDrosophila melanogaster.The homeotic mutation Polycomb (Pc ³) which partially transforms meso- and metathoracic legs into prothoracic legs did not significantly increase the frequencies of transdetermination in these leg dises and had clearly no effect on the direction of transdetermination (Table 1).We dedicate this publication to the memory of our teacher and advisor, the late Professor Ernst Hadorn, whose enthusiasm and interest stimulated our work     

Embryonic cAMP and developmental potential in Drosophila melanogaster

Summary Measurements of cAMP in early embryos of Drosophila melanogaster demonstrate that the dunce gene plays a major role, and the rutabaga gene a secondary role, in maternal regulation of embryonic cAMP content. Studying the double mutant combination, we find that variability in elevated cAMP content between individual embryos is associated with a wide variability in developmental potential. Embryos with about five times the normal cAMP content define a threshold between apparently normal and abnormal development. Measurements of cAMP content in anterior and posterior halves of embryos indicate that the posterior embryonic region, which is developmentally more sensitive to the effects of elevated cAMP than the anterior region, does not contain more cAMP than the anterior region. The variety of developmental defects observed is discussed in relation to possible targets of cAMP action.Offprint requests to: J.A. Kiger, Jr     

Ether-induced segmentation disturbances in Drosophila melanogaster

Summary Drosophila embryos, exposed to ether between 1 and 4 h after oviposition, develop defects ranging from the complete lack of segmentation to isolated gaps in single segments. Between these extremes are varying extents of incomplete and abnormal segmentation. On the basis of both their temporal and spatial characteristics, five major phenotype classes may be distinguished: headless — unsegmented or incompletely segmented anteriorly; gap — interruptions of segmentation not obviously periodic; alternating segment gaps — interruptions with double segment periodicities; fused segments; and short segments — truncations with single segment periodicities. Many defects resemble known mutant phenotypes. The disturbances in segmentation are predominantly global and frequently accompanied by alterations in segment specification, such that the segments obtained show no resemblance to the normal homologues. These features, together with the distinctive spatiotemporal characteristics of the defects, all point to segmentation as a dynamic process. The regular spacing of the segments and the fact that the entire range of defects is inducible by ether are further consistent with the hypothesis that at least part of the segmentation process may consist of physicochemical reactions coordinated over the whole body. The relationship between our data and data from genetic and other analyses are briefly discussed.     

The association between malathion resistance and acetylcholinesterase in Drosophila melanogaster

The relationship between the 50% survival time for flies feeding on a malathion-containing medium and the activity of acetylcholinesterase (AChE) was determined for 15 isofemale lines of Drosophila melanogaster. A significant correlation was found (r=0.28, P<0.05), with more resistant lines tending to have a lower level of AChE activity. An association between AChE and malathion resistance was also observed in a selection experiment. The AChE activity decreased in two of two populations selected for malathion resistance. AChE from these populations was altered in kinetic parameters (measured in crude head extracts) and electrophoretic mobility. Although the resistant AChE had a lower activity (V _m) on either a per milligram protein or a per individual basis, its apparent K _m for acetylthiocholine was lower than that of susceptible AChE. Recombination mapping of both low activity and fast electrophoretic mobility localized these traits to the region of the structural locus (Ace) on the third chromosome. The AChE activity of flies heterozygous for a variety of Ace lesions (kindly provided by Dr. W. M. Gelbart) was consistent with this location. The changes in AChE were suggested to have been caused by selection of alleles at the Ace locus.This work was supported by NSERC Grants A5857, G0183, and A0629.     

Basal relationships in the Drosophila melanogaster species group

The Drosophila melanogaster species group is a popular model for evolutionary studies due to its morphological and ecological diversity and its inclusion of the model species D. melanogaster. However, phylogenetic relationships among major lineages within this species group remain controversial. In this report, the phylogeny of 10 species representing each of the well-supported monophyletic clades in the melanogaster group was studied using the sequences of 14 loci that together comprise 9493 nucleotide positions. Combined Bayesian analysis using gene-specific substitution models produced a 100% credible set of two trees. In the strict consensus of these trees, the ananassae subgroup branches first in the melanogaster species group, followed by the montium subgroup. The remaining lineages form a monophyletic clade in which D. ficusphila and D. elegans branch first, followed by D. biarmipes, D. eugracilis, and the melanogaster subgroup. This strongly supported phylogeny resolves most basal relationships in the melanogaster species group, and provides a framework that can be extended in the future to encompass more species.     

Vestigial mutants of Drosophila melanogaster live better in the presence of aminopterin: increased level of dihydrofolate reductase in a mutant

Summary Vestigial (vg) mutants of Drosophila melanogaster are characterized by atrophied wings. In this paper we show that: (1) aminopterin an inhibitor of dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase induce nicks in the wings of wild-type flies and phenocopies of the vg mutant phenotype when vg/+ and vg ^B/+ flies are reared on these substances (vg^B is a deficiency of the vg locus). Only thymidine and thymidylate can rescue the flies from the effect of aminopterin. We propose that the vg phenotype is due to a decrease in the dTMP pool in the wings. (2) Mutant vg strains yield more offspring on medium containing aminopterin than on normal medium. The resistance of vg larvae to the inhibitor seems specific to the gene. This is the first case of aminopterin resistance in living eucaryotes. In contrast sensitivity of the vg larvae to FUdR is observed. (3) An increase in the activity and amount of DHFR is observed in mutant strains as compared with the wild-type flies.Our data suggest that the vg ⁺ gene is a regulatory gene acting on the DHFR gene or a structural gene involved in the same metabolic pathway.     

Second-site modifiers of the Delta wing phenotype in Drosophila melanogaster

Summary We have screened for dominant enhancers and suppressors of the wing phenotype associated with two Delta alleles: Dl ^9P39, an amorphic allele, and Dl ^FE32, an antimorphic allele. The interactions of some of the modifiers with Delta are due to haplo-insufficient expression of the corresponding genes. Although not explicitly shown for the remaining cases, we assume that haploin-sufficiency is also the basis for the relationships of these genes to Delta, since no allele specific interactions were observed. The modifiers found define 22 genes with pleiotropic expression, which can be classified into two groups: genes required for wing vein pattern formation and for neurogenesis, and genes which are not required for neurogenesis. Among the genes of the first group, Hairless and Star were previously known to participate in neural development. One further modifier was found which may correspond to a new neurogenic gene. The second group of genes is larger and includes already known loci, e.g., Plexate, blistered, plexus, etc, as well as other previously unidentified genes, which function during wing morphogenesis. Correspondence to: J.A. Campos-Ortega     

Vital genes in the heterochromatin of chromosomes 2 and 3 of Drosophila melanogaster

Heterochromatin has been traditionally regarded as a genomic wasteland, but in the last three decades extensive genetic and molecular studies have shown that this ubiquitous component of eukaryotic chromosomes may perform important biological functions. In D. melanogaster, about 30 genes that are essential for viability and/or fertility have been mapped to the heterochromatin of the major autosomes. Thus far, the known essential genes exhibit a peculiar molecular organization. They consist of single-copy exons, while their introns are comprised mainly of degenerate transposons. Moreover, about one hundred predicted genes that escaped previous genetic analyses have been associated with the proximal regions of chromosome arms but it remains to be determined how many of these genes are actually located within the heterochromatin. In this overview, we present available data on the mapping, molecular organization and function of known vital genes embedded in the heterochromatin of chromosomes 2 and 3. Repetitive loci, such as Responder and the ABO elements, which are also located in the heterochromatin of chromosome 2, are not discussed here because they have been reviewed in detail elsewhere.     

Genetic,ontogenetic, and tissue-specific variation of dipeptidases in Drosophila melanogaster

Three dipeptidases in Drosophila melanogaster are under independent genetic control and their structural genes have been localized, Dip-A to 2R and Dip-B and Dip-C to 3R (Voelker and Langley, 1978; Ohnishi and Voelker, 1981). These enzymes were characterized with respect to their substrate specificities, genetic variability (electrophoretic mobility and quantitative activity level), ontogeny (activity and isozyme pattern), and tissue localization. The dipeptide substrate specificities of DIP-A and DIP-B overlap each other considerably, but do not overlap with DIP-C. In natural populations, DIP-B and DIP-C are essentially monomorphic electrophoretically whereas DIP-A is polymorphic for three allozymes. Both DIP-A and DIP-B show quantitative genetic variation of activity level within an allozyme class. All three enzymes are expressed at all stages in the life cycle, but DIP-A and DIP-B activities vary considerably according to developmental stage and sex of adult. The tissue localizations of DIP-A and DIP-B activities show similar patterns and a nearly ubiquitous occurrence of both enzymes, but with particularly high values in larval and adult midguts and in the adult female reproductive system. These results suggest a general metabolic role for the enzymes, such as regulation of the concentrated pools of amino acids and oligopeptides found in Drosophila tissues.This work was supported by Public Health Service Grant GM 11546.Paper No. 7066 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh.     

The actions of the neonicotinoid imidacloprid on cholinergic neurons of Drosophila melanogaster

The neonicotinoid insecticide imidacloprid is an agonist on insect nicotinic acetylcholine receptors (nAChRs). We utilised fura-2-based calcium imaging to investigate the actions of imidacloprid on cultured GFP-tagged cholinergic neurons from the third instar larvae of the genetic model organism Drosophila melanogaster. We demonstrate dose-dependent increases in intracellular calcium ([Ca²⁺]_i) in cholinergic neurons upon application of imidacloprid (10 nM–100 μM) that are blocked by nAChR antagonists mecamylamine (10 μM) and α-bungarotoxin (α-BTX, 1 μM). When compared to other (untagged) neurons, cholinergic neurons respond to lower concentrations of imidacloprid (10–100 nM) and exhibit larger amplitude responses to higher (1–100 μM) concentrations of imidacloprid. Although imidacloprid acts via nAChRs, increases in [Ca²⁺]_i also involve voltage-gated calcium channels (VGCCs) in both groups of neurons. Thus, we demonstrate that cholinergic neurons express nAChRs that are highly sensitive to imidacloprid, and demonstrate a role for VGCCs in amplifying imidacloprid-induced increases in [Ca²⁺]_i.     

cell clones and pattern formation: Studies onsevenless,a mutant ofDrosophila melanogaster

Summary sev ^LY3,the only existing allele at thesev locus (1–33,2±0,2), behaves as strongly hypomorph or even as amorph. Ommatidia in asev compound eye have only seven receptor cells, the position of the R7 pattern element being vacant. Various criteria showing that the missing cell is R7 have been verified. These include (i) anatomical characteristics ofsev ommatidia; (ii) behaviour of central R cells insev rdgB double mutants; (iii) medullary projection of central R cell axons; and (iv) mitotic pattern ofsev imaginal discs. The analysis of morphogeneticsev-sev ⁺ mosaics has shown thatsev is expressed autonomously by R7 cells, indicating that thesev phenotype is not due to asev genotype of ommatidial pattern elements other than R7. The study of third instarsev imaginal discs has not brought any direct evidence for death of clustered presumptive R7 cells; however, clonal analysis of the developingsev compound eye has given evidence of developmental parameters comparable to those ofsev ⁺, therefore favouring the hypothesis that R7 cells die insev mutants. On the other hand,sev ⁺ seems to be required for the determination of the R7 cells, since thesev phenotype cannot be uncovered during the last mitoses of heterozygous mutant cells.     

Identification of a female-sterile mutation affecting yolk protein 2 in Drosophila melanogaster

Summary The three yolk proteins (YP1, YP2 and YP3) of Drosophila melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated in the developing oocytes to provide a nutrient source for embryogenesis. We have described the phenotype of a temperaturesensitive female-sterile mutant, fs(1) K313, and characterised its yolk proteins. This mutation affects the secretion of YP2 and is the first mutation affecting YP2 to be described. Using genetic and molecular tests we argue that the female-sterile phenotype results, at least in part, from the abnormal secretion of YP2 perturbing the follicle cell secretory pathway in general and thus causing defects in chorion protein secretion. The gene coding for YP2 in fs (1) K313 has been cloned and sequenced. Two amino acid substitutions have been found which probably cause the abnormal secretion of YP2 and the resulting female-sterile phenotype.     

Studies on transvection at the bithorax complex in Drosophila melanogaster

Summary We have studied the influence of some mutations in the bithorax complex on the observed synapsis dependent phenotype of the genotypes Cbx ¹Ubx¹/+ and bx ^34e/Ubx¹. The effect of these mutations is similar to that introduced by disruption of pairing or by the z ^a mutation. Among the bx mutations, we find that bx ⁸ behaves differently from most other bx mutations in its influence on the synapsis dependent phenotype. This observation induced us to map the position of bx ⁸ with respect to other bx mutations; we find that it maps between bx ^34e and bx ³. We show how some of the observations reported here can be fitted into a model of activation of the bithorax complex proposed by one of us.     

Heterosis in crosses between geographically separated populations of Drosophila melanogaster

Summary An experiment was performed to test the hypothesis that the genetic distance between populations estimated from enzyme loci could be used to predict the amount of heterosis that would occur in crosses between these populations. A partial diallel cross using 11 populations of Drosophila melanogaster from the AustralianPacific region and from England was carried out. Heterosis for larval viability, fecundity, cold shock mortality, and an index of these three traits was recorded. When two populations originating from the same location were crossed, no heterosis occurred, but otherwise heterosis was significant for all traits. For larval viability, a similar low level of heterosis occurred in all crosses. For cold shock mortality, the level of heterosis varied widely and fecundity showed a pattern intermediate between these two. The geographic distance between the sites from which populations originated was not correlated with the amount of heterosis in their crosses. There was a tendency for populations from ecologically different environments to show heterosis in crosses. Genetic distance based on ten enzyme loci was correlated with heterosis for cold shock mortality and the combined trait index. These results can be explained by the hypothesis that genes affecting larval viability are subject to strong, uniform selection in all populations, which limits the extent to which gene frequencies can drift apart. However, genes affecting cold shock mortality and the enzyme loci are subject to different selection pressures in different environments. This divergent selection combined with genetic drift causes divergence in gene frequency and heterosis.     

Nucleotide divergence of the rp49 gene region between Drosophila melanogaster and two species of the Obscura group of Drosophila

Summary A 2.1-kb SStI fragment including the rp49 gene and the 3 end of the -serendipity gene has been cloned and sequenced in Drosophila pseudoobscura. rp49 maps at region 62 on the tip of chromosome II of this species. Both the coding and flanking regions have been aligned and compared with those of D. subobscura. There is no evidence for heterogeneity in the rate of silent substitution between the rp49 coding region and the rate of substitutions in flanking regions, the overall silent divergence per site being 0.19. Noncoding regions also differ between both species by different insertions/deletions, some of which are related to repeated sequences. The rp49 region of D. pseudoobscura shows a strong codon bias similar to those of D. subobscura and D. melanogaster. Comparison of the rates of silent (K _S ) and nonsilent (K _a ) substitutions of the rp49 gene and other genes completely sequenced in D. pseudoobscura and D. melanogaster confirms previous results indicating that rp49 is evolving slowly both at silent and nonsilent sites. According to the data for the rp49 region, D. pseudoobscura and D. subobscura lineages would have diverged some 9 Myr ago, if one assumes a divergence time of 30 Myr for the melanogaster and obscura groups.Offprint requests to: C. Segarra